PROJECT SUMMARY/ABSTRACT Dopamine signaling in the striatum is critical for movement, yet the mechanistic basis for its permissive role in motor actions is incompletely understood. The longstanding view is that dopamine promotes movement by differentially modulating the striatum’s principal neurons, the D1 and D2 dopamine receptor expressing spiny projection neurons (SPNs). Specifically, striatal dopamine is thought to increase D1- and decrease D2-SPN excitability. This view has strong support from 1) ex vivo measurements of dopamine’s effects on D1- and D2- SPN excitability and the in vivo observations that 2) ablating dopamine neurons decreases D1- and increases D2-SPN activity and 3) the selective activation of D1- or D2-SPNs respectively promotes or suppresses movement. However, several recent findings from in vivo recordings of D1- and D2-SPN activity do not support a simple “go/no-go” model for D1- and D2-SPN function in movement. Specifically, in vivo recordings have shown that D1- and D2-SPNs co-activate in spatially overlapped clusters, both increase their activity at higher running speeds, and both decrease their activity at motion offset. Therefore, it remains unclear precisely which aspects of D1- and D2 activity (e.g., levels, timing, or spatial coordination) are modulated by dopamine signaling and how this promotes movement. To address these questions, we have developed three, multiphoton imaging approaches to simultaneously record 1) D1- and D2-SPN activity, 2) dopamine axon activity and D1- or D2-SPN activity, and 3) immediate early gene expression tagging and D1- or D2-SPN activity in vivo. We will use these tools to image neural activity during training in a dopamine-dependent, conditioned-avoidance motor learning task. Our preliminary data indicate that dopamine is released during learned movement in this task, and that D1- and D2-SPNs encode these movements with different levels, timing, and spatial coordination. We hypothesize that these changes are necessary for motor learning and result from dopamine’s gradual and differential effects on the strength of excitatory synaptic connections in specific subsets of D1- and D2-SPNs. We will test this hypothesis by integrating the results from our in vivo imaging experiments with ex vivo measurements of synaptic strength in D1- and D2-SPNs. Overall, our experiments have the potential to resolve a central conflict in our understanding of dopamine’s role in motor control and how this process goes awry in neurological and psychiatric disease.